1. Field of the Invention
The present invention relates to a method of removing an Fmoc (9-fluorenylmethoxycarbonyl) group, and a production method of a peptide by using said method.
2. Discussion of the Background
Fmoc group is an important protecting group of amino acid and an amino group of a peptide in the peptide synthesis. As a result of this removal of the Fmoc group, dibenzofulvene (DBF) or DBF derivative is produced as a byproduct. Fmoc group is generally removed by using a base. For example, when Fmoc group is removed by using an amine, an adduct of DBF and amine (hereinafter sometimes to be abbreviated as a “DBF-amine adduct”) is by-produced as a DBF derivative. When the peptide synthesis is continued while DBF or DBF derivative still remains, a side reaction such as 9-fluorenylmethylation occurs. Thus, it is desirable to remove DBF or DBF derivative efficiently. In addition, when amine is used to remove Fmoc group and DBF in the peptide synthesis, the amine needs to be removed before the next condensation step.
Non-patent document 1 describes a method for removal of a DBF-amine adduct in the liquid phase peptide synthesis, which includes adding a hydrocarbon solvent such as hexane and the like for trituration of a residue obtained by concentrating a reaction extract to dryness, thereby dissolving a DBF-amine adduct in the solvent, and isolating the deprotected peptide as crystals. However, this method is poor in operability, sometimes fails to reproduce at a large scale, and is unsuitable for industrial production. In addition, when a desired deprotected peptide is an oily substance, this method cannot be used. Furthermore, the method is associated with problems of low recovery rate and the like due to dissolution of peptide itself in a hydrocarbon solvent when the peptide chain is short.
To solve the problem mentioned above, patent document 1 describes a method including stirring a reaction mixture after removal of the Fmoc group in a hydrocarbon solvent and a polar organic solvent, separating the hydrocarbon solvent layer and the polar organic solvent layer, and removing the hydrocarbon solvent layer in which a DBF and/or DBF-amine adduct is dissolved. Patent document 2 describes a method including contacting a reaction mixture containing a DBF-amine adduct with carbon dioxide to form a carbonate of a DBF-amine adduct, and removing the carbonate. However, both patent documents 1 and 2 do not describe use of sulfanyl group (SH)-containing fatty acid for the removal of an Fmoc group.
On the other hand, non-patent document 2 describes a method including using 1-octanethiol and the like as scavengers of DBF produced during removal of Fmoc group. In this method, an adduct of DBF and thiol (hereinafter sometimes to be abbreviated as a “DBF-thiol adduct”) is produced. In an experiment using 1-octanethiol in non-patent document 2, (a) trituration or (b) trituration and recrystallization was performed in the same manner as in the method of non-patent document 1 to remove a byproduced DBF-thiol adduct (Table 1), and the operability is poor.
Patent document 3 describes a method including using an amine containing free anion or potential anion, or a thiol containing free anion or potential anion, as a scavenger to remove a residual carboxy component in the peptide synthesis comprising reacting an excess amount of the carboxy component with an amino component. Patent document 3 further describes, “the aforementioned scavengers are also used for the deprotection of a peptide under elongation”. However, in the method of patent document 3, the aforementioned scavengers (thiol containing free anion or potential anion etc.) are used to deprotect peptide to perform removal of a temporal protecting group contained in the free anion (scavenger), and deprotection of the N-terminal of peptide under elongation by a single treatment. Patent document 3 does not describe use of the aforementioned scavengers for removing Fmoc group or trapping DBF.
Patent document 4 describes a method of producing a compound represented by Fm—S—X—COOH (wherein Fm is 9-fluorenylmethyl), comprising reacting a compound represented by HS—X—COOH (wherein X is an alkylene chain having 1-5 carbon atoms) with a compound represented by Fm—R1 (wherein Fm is 9-fluorenylmethyl, and R1 is a chlorine atom and the like), or Fmoc-R2 (wherein Fmoc is 9-fluorenylmethoxycarbonyl, and R2 is succinimidyloxy and the like). However, patent document 4 relates to a production method of Fm—S—X—COOH, and does not relate to the deprotection of an amino group-containing compound protected by an Fmoc group. Patent document 4 proposes using an inorganic base since the above-mentioned reaction using an organic base affords HS—X—COOH only in a low yield.